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Tuesday, July 17, 2012

There has been a lot of interest in my Zombie Protocol series so far, and for that I want to thank everyone who has wandered into the Dark Laboratory (even if you were just looking for the little girls' room). Zombies are always hot around Comic Con, and there is always some new book or movie coming out. It's easy to find pop culture information on the next apocalypse, but much harder to find scientific discussions about the phenomenon (hence, this blog!).

Tara Smith had a nice piece yesterday in Science Blogs about teaching children science concepts using zombies as a backdrop (here's the link). Disease transmission, pandemics, zombie bugs, neurobiology, and other biological topics were used to discuss what strategies the kids would use for different types of zombie outbreaks. They also used watermelons as, um, volunteers, to compare the effectiveness of various weapons. Where were these people when I was in school!!

Coolest logo EVER!

Anyhow, I'm also starting to include links on the right to various sources of zombie science. Scott Kenemore's blog is doubly good as it also discusses new zombie books and movies. He's also on the advisory board of the Zombie Research Society, which looks a little tongue-in-cheek but has some heavy hitter scientists on their Board. Coincidentally, Tara Smith is also on the Advisory Board. Both sites have an occasional discussion about scientific literature and are worth a read. If you know of other sites that have a scientific bent to zombie research, or any other topic that might be of interest to the Lab, please leave a note below. Spam will, of course, be fed to the zombies...

Wednesday, July 11, 2012

With 'bath salts' in the news these days, there is suddenly a lot of interest in zombification. If you recall, in part 1 of the Zombie Protocol, I described the role of tetrodotoxin in the creation of Haitian zombies. There are, however, other ingredients in the zombie poison that have significant pharmacological activity. Besides related TTX toxins from other species of fish, there can also be poisons from frogs, reptiles, and spiders. The poison from the frog, Bufo marinus, is of particular interest as it contains bufotenine (5-hydroxy-N,N-dimethyltriptamine, or 5-OH-DMT). Bufotenine is an alkaloid related to DMT and 5-MeO-DMT, as well as psilocin (one of the psychedelic molecules found in mushrooms). It is also related to serotonin, a well-known neurotransmitter.

Serotonin: Important neurotransmitter

Therefore, it seems very plausible that bufotenine is present in the zombie powder to enhance the effect of the tetrodotoxin. Remember from my last post that TTX is present at sub-lethal doses (in some studies, the amount of TTX is very low -- see Benedek and Rivier, Toxicon 27:473-480, 1989), so other components of the powder could be necessary to boost the effect of the mixture without killing the victim. Psychoactive substances would also be very effective since the victim is likely to be conscious while under the influence of TTX. I mentioned the Poe-like experience of being buried alive without any way to move or scream... now imagine that happening while having horrible hallucinations! You talk about a bad trip!!

Seed pods from Mucuna pruriens (Wikapedia)

Plants known to be irritants were also added to some of the zombie powders. In Wade's paper (abstract), he lists Urera baccifera, Dalechampia scandens, and Mucuna pruriens as some of the more common plants. These plants all have irritating hairs or needles that cause contact dermatitis. In the case of M. pruriens, the hairs on the seed pods contain (interestingly enough) serotonin. Why would irritants be important to a zombie poison? Personally, I think this is one of the ingenious parts of the protocol... the irritants are there for delivery. To pull off the zombification, a voodoo priest would have to administer this part of the poison without the victim's knowledge. Poisoning food or using darts are quite tricky, but what about a chance encounter on the busy streets of Port-au-Prince? You have the drug cocktail smeared on the end of a walking stick, or some other convenient device. Following your target through the busy streets, you wait until you have a good opportunity. He's distracted by a street vendor, so you bring the stick high and as you pass behind him, you gently poke him in the back of the neck. He may not even notice but after you have left, he feels a burning sensation and scratches it. The itching and burning intensifies until he scratches it raw. The drugs are now able to enter the blood and begin the process of poisoning the victim. He won't remember the chance encounter, but you know he will be at the hospital soon, and at the morgue not too long after that.

I know I promised zombie cucumbers in Part 2... but they will show up in Part 3, I promise.

Friday, July 6, 2012

It's been bat shit crazy in the Not-So-Dark Laboratory (otherwise known as my day job), so my apologies for getting behind on the real crazy science. One of my first posts (zombie science) talked about zombie science and the potential role of Toxoplasma gondii. T. gondii is a neurotropic protozoan parasite that has been linked to a variety of mental disorders. When rats are infected, they lose many of the behavioral adaptations that protect them from cats and aggressively try to get themselves eaten. Sadly, the effect of T. gondii on humans seems more subtle. But there is a new paper out on the relationship between T. gondii and suicide, which provides stronger evidence that this parasite is a potentially serious global problem.

Several recent studies have shown that T. gondii affects human behavior. A meta analysis published in 2007 (abstract) found that there is a correlation between schizophrenia and serum antibody levels against T. gondii. The predictive power of the association was weak, since more that one third of the entire population is thought to be seropositive for T. gondii, and aside from a few ex-girlfriends and that driver on the 15 the other day, not all of them are crazy. One flaw with the meta-analysis is that it was not clear when the patients were infected relative to the onset of disease. If they all got T. gondii after they became schizophrenic, then the association is meaningless. The new study just published by Pedersen et al. (abstract) tries to account for that by measuring T. gondii antibody levels when women gave birth (using samples from heel-stick cards in the birth records) and then looking at the risk of depression and suicide later in their life. Seropositivity was 26.8% at delivery, consistent with the notion that 1/3 of the population has already been infected (remember that infants don't start making their own antibodies for about three months after birth, so these are the mom's antibodies).

From Pedersen et al, Arch. Gen. Psych. 2012.

Pedersen et al. found that seropositive women had a 1.53-fold greater risk of self-directed violence (ie, suicide) than seronegative. Women with the highest antibody titers had nearly a 2-fold higher risk. The risk is small, but significant and is also consistent with other recent studies on the relationship between T. gondii antibody titers and mental illness (for example, see this). I'm generally not a big fan of these types of analyses, because the data could simply be a case of "true, true, and unrelated". There is no mechanistic hypothesis for why antibodies against T. gondii would alter behavior, or whether the infection caused permanent damage to the brain. However, evidence continues to accumulate that T. gondii infection can cause permanent changes in human behavior, and with billions of people having been infected at some point, it isn't too hard to see the beginnings of a zombie apocalypse.

Tuesday, April 17, 2012

As I mentioned in my previous post, some reports of zombies are more likely observations of people suffering from mental illness, long-term drug abuse, or both. Given the loose definition of a zombie as somebody "acting strange" who had been missing and presumed dead, one wonders how many urban dwellers and street people in this country might be considered zombies. I was at a conference in Barbados a few years back and walked past a homeless guy on Broad St. in Bridgetown. He rose out of a pile of belongings to approach me, his legs stiff and his arms trembling. He was a pitiful sight, and communicated using grunts and various hand gestures. He wound up following me for a block and even after I crossed the street and doubled back, he was still behind me. I told everyone back home that I had seen a zombie, no doubt fulfilling my obligation to keep these legends alive.

But what about "real" zombies? Is there a way to bring someone back from the dead? Does the Zombie Protocol exist? Without a doubt, the answer to this is 'yes'. Although the legend of the zombie has existed in Haiti for generations, there is enough physical evidence to suggest that the process of zombification is likely rooted in scientific fact. Please ignore the Hollywood ideas of brain-sucking zombies, and think instead about a circus act. How would somebody make a zombie without learning all that dark magic (which, by all accounts, is very difficult)? You fake it, but you fake it well.

Tetrodotoxin structure: Note the guanidido group (NH2+)
group on the left.

In Wade Davis' excellent article The Ethnobiology of the Haitian Zombi (abstract), he lists the composition of zombie poisons from five separate locations around Haiti. Although there were many differences, based on what was available at the different geographical locations, there were several key ingredients. One of the most important was the puffer fish (Diodon hystrix, Diodon holacanthus, and others). Puffer fish in the region have very high levels of tetrodotoxin (TTX), which is, of course, the same toxin found in Japanese fugu fish. Tetrodotoxins are extremely toxic molecules and act by blocking Na ion channels, presumably through the positively charged guanidido group, which competes for the Na binding site in the channel. Without Na exchange, nerve impulses do not propagate which leads to paralysis, as well as cardiac and respiratory failure. The LD50 of TTX is estimated to be about 5 ug/kg in humans (less than1 mg of TTX), so what if a person ingested something a little less than that? Respiratory function decreases, blood pressure drops to near zero, pupils are fixed and dilated, the body is paralyzed, but maybe, just maybe, they won't actually die. This is the first trick of the Voodoo priests, namely, to make the family think the victim is dead by titrating the amount of puffer fish in the poison to near the LD50.
What is interesting, and also horrifying, is that TTX does not usually affect cognitive function. This means that the victim falls ill and becomes "dead" while remaining conscious. They hear the doctors discussing the death, hear the family wailing in grief, hear the preparations for burial, and then find themselves in a box, quiet and still. Imagine the terror of being buried alive but having no way to move, scream, or otherwise respond to the panic. The fear is completely trapped in the victim's mind.. Poe couldn't have scripted that much better...

Wednesday, March 28, 2012

While I was doing research for my first book, I read a lot of articles on zombies. Not the brain-eating type that are often found in movies, but real reports of zombies. It was sometimes difficult to differentiate a true case from an urban legend ("I swear I saw a zombie walk right into that bar!") or a case of mistaken identity ("I thought it was a zombie, but it turned out to be just my mother-in-law..."). However, I ran across a very interesting article published in 1997 in the Lancet, a highly respected medical journal, on clinical findings from several reported zombies (here's the reference... a search will turn up the pdf elsewhere). The paper, published by Roland Littlewood, from University College, London and Chavannes Douyon, a Haitian doctor, makes a fairly shocking claim. Although the process of zombification has been attributed to both poisoning or sorcery, the empirical data from Haiti suggest that people demonstrating behaviors consistent with a zombie are on the order of a thousand cases per year. This is truly staggering, if accurate.

The Lancet article studies three reported cases of zombies during 1996-1997. Patients FI and WD, although described by relatives as becoming ill and dying prior to their return months or even years later, were probably not dead to begin with. Both patients suffered from mental illness and it seems more likely that they had simply wandered away from their homes and were taken in by other families. For FI, the Lancet study concluded that she suffered from catatonic schizophrenia, a syndrome that could be considered zombie-like by locals. WD likely suffered from organic brain syndrome and epilepsy. The Lancet article speculated that these conditions were due to an unspecified period of anoxia. Perhaps WD had been buried alive? Unlike FI's coffin, which was filled with stones, WD's family refused to open the grave to Littlewood and Douyon.

Patient MM was a 31-year old female who had disappeared at age 18 after attending a service for a friend who had been zombified. She fell ill shortly after and died. Thirteen years later, she reappeared in the town and claimed to have been a zombie slave at a distant farm. Littlewood and Douyon found her to be of very low intelligence but were unable to diagnose a more specific mental illness. Upon return to the town near her captivity, locals recognized her as a zombie and several people argued over their claim to her. Interestingly, she had a round, 1cm scar on her back, similar to a scar found on patient WD. What were these scars from?

Unfortunately, they ultimately state that none of the three were true zombies, and that much of their behavior could be explained by mental illness. However, one of the references they cite is the now classic paper by Wade Davis, published in the Journal of Ethnopharmacology in 1983, titled "The ethnobiology of the Haitian zombi" (abstract). Is that a cool title or what! In this little gem, he provides a pretty detailed 'zombie protocol' and this forms the basis for Davis' book The Serpent and the Rainbow. In my next post, I'll discuss his findings and several other classic zombie manuscripts...

Wednesday, February 8, 2012

Just when you thought the zombie apocalypse couldn't get any worse... now we have roaches to worry about! Fortunately, zombie roaches in the wild are not aggressive, brain-sucking insects. It turns out that the wasps that make the zombies are what we should really be concerned about.

Jewel Wasp (A. compressa)
Image courtesy of Morphbank.net

The Jewel Wasp (Ampulex compressa) has an elegant and extremely sophisticated method to secure a good home for its eggs. It attacks and injects a cocktail of neurotransmitters into the brain of a roach, effectively turning it into the slave of the wasp (Educated Earth has a good video of this process, see here). The wasp then drags the roach to a suitable location and lays its eggs in the abdomen. The roach is hypokinetic for several days until the larvae hatch and then they slowly consume the host. Interestingly, the larvae eat the internal organs in a way that maximizes the lifespan of the roach. It's a morbid process, but how the wasp achieves this zombification is truly incredible.

The wasp actually stings the roach twice. The first sting is to the thorax and produces a mild and very transient anesthetic that paralyzes the front legs. A study by Moore et al. (abstract) showed that the active components of the first sting activate GABA receptors (GABA itself, and receptor agonists beta-alanine and taurine). During the minute or so of action, the wasp then stings the roach again, but in a very precise location within the brain. In a sense, the wasp is acting like a brain surgeon, who needs their patient to remain still while a precise operation is done. This second injection is a cocktail of neuroactive compounds that have very specific effects on the roach's brain. After the anesthetic wears off, the roach grooms itself excessively and then becomes lethargic. It is not paralyzed, just sluggish. When the first studies on A. compressa were done, there was a great deal of controversy over whether the venom was delivered to the central nervous system, or directly to the site of action in the brain. In an elegant study by Haspel et al. (abstract), they milked the venom from wasps and injected them with radiolabeled carbon (14C). This meant that the fresh venom produced by the wasp would contain the radiolabel. Haspel could then visualize the location of the venom in the roach brain by taking pictures of brain slices. Those of you who think that would be cool as hell, raise your hand!!

Anyhow, it was found that the neurotoxin attacks the brain directly, with the most venom at the supra-esophageal ganglia (SupEG) and the sub-esophageal ganglia (SEG). In insects, these areas are thought to provide "higher order" brain function. A recent study by Gal et al.(abstract) found that the venom affects the drive to initiate and maintain walking. Other motor skills (like flying, swimming, self-righting) were not affected. Based on the electrochemical difference between stung and unstung wasps, they concluded that stung wasps had a deficit in the ability to "reach a decision" to walk. There must be human versions of this wasp because I swear, there are days when I have trouble reaching the decision to get off the couch.

A follow-up study by Gal et al in 2010 (PLoS article) made another very interesting discovery. Roaches who had their brain ganglia removed were stung for a much longer period of time (196 sec) than normal roaches (39 sec). Why? Because the wasp was actively seeking this part of the brain and couldn't find it! What does it search for? A specific morphology? A defined depth within the brain? Nobody knows. Furthermore, why is it so important for the wasp to target this region so specifically? The answer is also still open to speculation but this has zombie written all over it. The wasp needs the roach to be compliant to move it into the proper position for incubation of the larvae, but resist the urge to flee for several days. So, the venom basically takes away whatever "freewill" the roach might have had.

Octopamine
(image courtesy of Wikidocs)

So, what is this supernatural cocktail? Not a whole lot is known about the composition of the venom (another great project for biohackers or kids looking for obscure science fair projects). I will post more details later but one key component appears to be an antagonist of the octopamine receptor. Octopamine is a monoamine neurotransmitter that is specific to invertebrates and OA levels are known to be associated with the hypokinetic state. Several years ago, Rosenberg et al. (abstract) gave roaches a octopamine receptor agonist and found that motor activity in stung subjects was at least partially restored. Other receptor agonists and antagonists had little effect. This is certainly one clue about how A. compressa makes zombie cockroaches but for now, the rest of the process is all voodoo.

Tuesday, January 17, 2012

Resveratrol is in the news again but this time for all the wrong reasons. No, it didn't just break up with a pop star or get busted for breaking probation. Stunning allegations from the University of Connecticut suggest that a significant percentage of Dipak Das' (UConn professor and Director of the Cardiovascular Research Center) scientific research on RES may suffer from scientific fraud (see here for just one article). I looked at some of the (60,000 page!) report and it looks like much of the fraud was based on images of Western blots that had been altered or fabricated. I have long complained about figures of Western blots in various publications where only the bands of interest are shown with no molecular weight markers or anything. This is like buying a car based on a picture you see on the internet. Yes, there is a band there but you have no idea how good the antibody is that you are using to probe with, if the protein runs at the right place on the gel, etc. In Das' case, it looks like random bands were just pasted on there. No bueno, pal, no bueno.

Does this mean that resveratrol is now demoted to a worthless contamination in an otherwise tasty glass of wine? Um, no. I'll admit I have read some of Das' stuff and it has influenced some of my opinions about RES, but there are a whole host of researchers out there that have demonstrated how RES impacts biological pathways and (in my opinion) there is very clear evidence that it has a significant effect if the dose is high enough. Unfortunately, cases like this place a stigma on research involving RES and could hinder progress towards understanding the physiological benefit of this molecule.

Ok, so now on to better news. My favorite wine goddess maker, Kerith Overstreet from Bruliam Wines, has a new blog post on the cardioprotective properties of wine. It's pretty funny (you can check it out here) but in it she highlights not RES, but oligomeric proanthocyanidins (OPCs). If you recall an early post I made on the magic of winemaking (here), you may recognize OPC as another term for polymerized flavinoids, which include tannins such as catechin. Most OPCs originate from the grape skin, so the amount of OPC in any given bottle can vary dramatically. The final levels depend, in part, on how long the grape skins are left in the fermentation since it is the rising alcohol content that ultimately extracts the monomeric proanthocyanidins from the grape. Therefore, craft is a big variable in determining the benefit of wine to the drinker (we are all counting on you, Kerith!) Interestingly, Das was involved in a company called Dry Creek Nutrition, that was trying to purify and sell proanthocyanidins. In light of the Das debacle, maybe OPCs are the new RES!